| Background:Esophageal cancer（EC）ranks among the most prevalent gastrointestinal malignancies,exhibiting the highest incidence rates in China.The majority of these cases involve histological types classified as esophageal squamous cell carcinoma（ESCC）.Currently,treatment for esophageal cancer primarily consists of surgery,chemotherapy and radiotherapy;however,long-term outcomes remain unsatisfactory,posing a significant threat to public health.In recent years,immunotherapy targeting immune checkpoints has achieved breakthroughs in treating solid tumors,revolutionizing the management of numerous malignancies,including ESCC.Several pertinent clinical studies have also corroborated the safety and efficacy of neoadjuvant immunochemotherapy for ESCC.Regrettably,some patients experience little to no benefit from immunotherapy,with the tumor microenvironment playing a crucial role in this limited efficacy.Our study aims to examine the role of SYNGR2 in the tumor microenvironment of esophageal squamous carcinoma by employing bioinformatics data and methodologies.Methods:We summarized the clinical data of patients diagnosed with ESCC and treated with neoadjuvant immunochemotherapy between September 2020 and December 2022.Treatment efficacy was evaluated based on RECIST Ver1.1 criteria and surgical specimens to assess tumor regression grading.Fluorescent multiplex immunohistochemistry was employed to detect and evaluate the molecular expression of T cells,B cells,macrophages,and NK cell-related indicators,as well as tertiary lymphoid structures in tissue samples.We observed changes in the tumor immune microenvironment before and after neoadjuvant therapy.Sequencing data and clinical data for ESCC patients were obtained from the TCGA and GEO databases to analyze SYNGR2 expression in ESCC tissues and normal esophageal tissues.Kaplan-Meier curves assessed the prognostic value of SYNGR2 in patient survival,while ROC curves and nomograms evaluated its diagnostic value for ESCC.Univariate and multifactorial regression analyses explored independent variables affecting ESCC clinical prognosis.Subsequently,the STRING database was used to construct a protein interaction network,and genes closely associated with SYNGR2 and ESCC prognosis were identified through GO and KEGG enrichment analyses.Finally,immune infiltration analysis was performed on ESCC samples using single-sample gene set enrichment analysis.The relationship between ESCC and immune cell subpopulations was determined via Spearman’s correlation coefficient.The correlation between SYNGR2 gene expression and the abundance of tumor-infiltrating immune cells and gene markers for these cells was analyzed using the TIMER and TISIDB databases.Result:（1）Between September 2020 and December 2022,69 patients with ESCC underwent neoadjuvant immunochemotherapy.Solid tumor responses were evaluated as follows:4 patients（5.8%）exhibited complete response（CR）,34 patients（49.3%）showed partial response（PR）,29 patients（42.0%）demonstrated stable disease（SD）,and 2 patients（2.9%）had progressive disease（PD）.The objective response rate（ORR）was 55.1%.Thirty-six surgeries were completed,with 12patients（33.3%）achieving tumor regression grade TRG1（p CR）,13 patients（36.1%）attaining TRG2（MPR）,and a combined total of 11 patients（30.6%）reaching TRG3and TRG4.（2）Results from the fluorescent multiplex immunohistochemistry test indicated significant changes in the tumor microenvironment of ESCC patients with varying immune responses.These changes were primarily characterized by the infiltration of tumor-infiltrating lymphocytes and the formation or maturation of tertiary lymphoid structures.The latter was associated with improved immune responses in esophageal squamous cell carcinoma patients.（3）Analysis of results from the TCGA database revealed that SYNGR2expression levels are significantly elevated in esophageal squamous cell carcinoma.This finding was further validated using the GEO database（GSE26886,GSE45670,GSE161533,p<0.001）.Univariate and multifactorial regression analyses of SYNGR2 expression levels in different ESCC patients from the TCGA database demonstrated that SYNGR2 overexpression is significantly associated with poorer T-stage（T3 and T4 compared to T1 and T2,p=0.034）and overall survival（p=0.031）.SYNGR2 expression level is an independent prognostic factor for overall survival in ESCC.ESCC patients in the TCGA database were divided into two groups with high and low SYNGR2 expression.Kaplan-Meier survival analysis results indicated that SYNGR2 expression level is significantly associated with overall survival（HR=4.34,p=0.002）and disease-specific survival（HR=3.64,p=0.015）in ESCC patients.Under the ROC curve（AUC）of 0.881（CI:0.778–0.984）,time-dependent survival ROC curves were created to predict 1-,3-,and 5-year survival rates.The AUC values of these curves（0.573,0.812,and 0.697）were greater than 0.55,indicating that SYNGR2 expression level has diagnostic value for ESCC patients.Construction of the nomogram prediction model plot revealed that SYNGR2expression level has a higher predictive value for ESCC patient prognosis than clinical characteristics such as age,T stage,M stage,and histological grading.The STRING database was used to construct a protein interaction network,and 38 key genes closely associated with SYNGR2 and ESCC prognosis were identified through GO and KEGG enrichment analyses.These key genes were strongly associated with the protein ubiquitination process（p=2.29×10–4）,SYNGR2 expression levels were negatively correlated with the level of helper T cell infiltration（r=-0.264,p=0.017）,SYNGR2 methylation was associated with chemokines CCL2(r=0.404,p<1.6×10–8)and CXCL12(r=0.393,p<4.04×10–8),chemokine receptor CCR1(r=0.321,p<9.65×10–6)and CCR2(r=0.337,p<3.21×10–14).In contrast,immunosuppressants CXCL12(r=0.393,p<4.04×10–8)and human tumor necrosis factor receptor superfamily member 4(TNFRSF4,r=0.338,p<3×10–6)and colony-stimulating factor 1 receptor(CSF1R,r=0.395,p<3.5×10–8)and human cell programmed death protein 1 ligand 2(PDCD1LG2,r=0.448,p<1.46×10–6)expressions were positively correlated.Conclusion:The tumor microenvironment undergoes changes following neoadjuvant immunochemotherapy in patients with ESCC,with the formation or maturation of tertiary lymphoid structures being closely associated with improved immune responses.SYNGR2 overexpression is significantly linked to prognosis and immune cell infiltration within the tumor microenvironment of patients with ESCC.As such,it holds potential as a prognostic and immune response biomarker for ESCC. |
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